Cigarette smoking causes 80-90% of all cases of chronic obstructive pulmonary disease, COPD, which annually kills nearly 125,000 people in the United States and costs this country $37.2 billion. Cigarette smoke rapidly increases the permeability of airway epithelium, especially in those with COPD. The barrier function of an intact epithelium is maintained by tight junctions between adjacent cells. Little is known about the mechanisms underlying tight junction function in respiratory epithelium. Our long-range goal is to define the mechanisms responsible for cigarette smoke induced alterations in lung epithelial cell function in order to develop effective therapeutic strategies to inhibit this altered function. The objective of this proposal is to refine the methodology of in vitro air-liquid interface exposure of lung epithelial cells to whole, fresh cigarette smoke to improve the identification of regulatory mechanisms controlling smoke altered tight junction function. The central hypothesis of this proposal is that smoke from current market relevant cigarettes alters tight junction function because of altered cytoskeletal regulation by Rho kinase (ROCK) and myosin light chain kinase (MLCK). This hypothesis is based on strong preliminary findings obtained using a first generation in vitro model system. The rationale for this proposal is that the development of an improved in vitro exposure system that more closely reflects the current in vivo condition in smokers will allow mechanisms that control lung epithelial cell function to be defined, resulting in the identification of potential targets for therapeutic interventions. The central hypothesis will be tested and the objective accomplished through two specific aims: 1) refine an in vitro air-liquid interface exposure system that models in vivo lung exposure; 2) determine the extent to which ROCK and MLCK contribute to the reversible loss of tight junction function in lung epithelial cells exposed to cigarette smoke. The proposed work is innovative because it builds on a recently developed technique to expose lung epithelial cells in vitro under conditions that mimic the exposure conditions within the in vivo lung. Our expectation is that this approach will establish that ROCK and MLCK through their regulation of myosin contribute to the regulation of tight junction function in lung epithelial cells. The outcome is significant because it will begin to define the signal transduction pathways that regulate cigarette smoke induced loss of tight junction function in lung epithelium, and will provide insights for the development of strategies to control the loss of epithelial integrity. The proposed study fulfills the AREA program objectives of meritorious research to engage students in the research endeavor and to improve the research environment at this primarily undergraduate institution. PROJECT NARRATIVE. Cigarette smoking causes chronic obstructive pulmonary disease (COPD) which annually kills nearly 125,000 people in the US and costs this country $37.2 billion. Cigarette smoke causes the lungs in people to become leaky, especially in those with COPD. This project will begin to address how cigarette smoke makes lung cells leaky. [unreadable] [unreadable] [unreadable]